IRLF 


AGRIC. 
UBRART 


STUDIES  ON  CLUBROOT  OF 
CRUCIFEROUS  PLANTS 


A   THESIS 

PRESENTED  TO  THE  FACULTY  OF  THE  GRADUATE  SCHOOL 
OF  CORNELL  UNIVERSITY  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 


BY 

CHARLES    CHUPP 


Published  as  N.  Y.  (Cornell)  Agr.  Exp.  Sta.  Bui.  387,  1917. 


STUDIES  ON  CLUBROOT  OF 
CRUCIFEROUS  PLANTS 


A   THESIS 

PRESENTED  TO  THE  FACULTY  OF  THE  GRADUATE  SCHOOL 
OF  CORNELL  UNIVERSITY  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 


BY 

CHARLES    CHUPP 

\\ 


Published  as  N.  Y.  (Cornell)  Agr.  Exp.  Sta.  Bui.  387,  1917. 


'.  *, 


MARCH,   1917  ^    •'•'a*  ''/^BULLETIN  387 

< 

,,  CORNELL  UNIVERSITY 

AGRICULTURAL  EXPERIMENT  STATION 


STUDIES  ON  CLUBROOT  OF   CRUCIFEROUS 

PLANTS 


CHARLES    CHUPP 


ITHACA,  NEW  YORK 
PUBLISHED  BY  THE  UNIVERSITY 


AGRICULTURAL  EXPERIMENT  STATION 

MKL 


ALBERT  R.  MANN,  B.S.A.,  A.M.,  Acting  Director. 

HENRY  H.  WING,  M.S.  in  Agr.,  Animal  Husbandry. 

T.  LYTTLETON  LYON,  Ph.D.,  Soil  Technology. 

JOHN  L.  STONE,  B.Agr.,  Farm  Practice. 

JAMES  E.  RICE,  B.S.A.,  Poultry  Husbandry. 

GEORGE  W.  CAVANAUGH,  B.S.,  Agricultural  Chemistry. 

HERBERT  H.  WHETZEL,  M.A.,  Plant  Pathology. 

ELMER  O.  FIPPIN,  B.S.A.,  Soil  Technology. 

G.  F.  WARREN,  Ph.D.,  Farm  Management. 

WILLIAM  A.  STOCKING,  JR.,  M.S.A.,  Dairy  Industry. 

WILFORD  M.  WILSON,  M.D.,  Meteorology. 

RALPH  S.  HOSMER,  B.A.S.,  M.F.,  Forestry. 

JAMES  G.  NEEDHAM,  Ph.D.,  Entomology  and  Limnology. 

ROLLINS  A.  EMERSON,  D.Sc.,  Plant  Breeding. 

HARRY  H.  LOVE,  Ph.D.,  Plant  Breeding. 

ARTHUR  W.  GILBERT,  Ph.D.,  Plant  Breeding. 

DONALD  REDDICK,  Ph.D.,  Plant  Pathology. 

EDWARD  G.  MONTGOMERY,  M.A.,  Farm  Crops. 

WILLIAM  A.  RILEY,  Ph.D.,  Entomology. 

MERRITT  W.  HARPER,  M.S.,  Animal  Husbandry. 

JAMES  A.  BIZZELL,  Ph.D.,  Soil  Technology. 

GLENN  W.  HERRICK,  B.S.A.,  Economic  Entomology. 

HOWARD  W.  RILEY,  M.E.,  Farm  Mechanics. 

CYRUS  R.  CROSBY,  A.B.,  Entomology. 

HAROLD  E.  ROSS,  M.S.A.,  Dairy  Industry. 

KARL  McK.  WIEGAND,  Ph.D.,  Botany. 

EDWARD  A.  WHITE,  B.S.,  Floriculture. 

WILLIAM  H.  CHANDLER,  Ph.D.,  iWology. 

ELMER  S.  SAVAGE,  M.S.A.,  Ph.D.,  Animal  Husbandry. 

LEWIS  KNUDSON,  Ph.D.,  Plant  Physiology. 

KENNETH  C.  LIVERMORE,  Ph.D.,  Farm  Management. 

ALVIN  C.  BEAL,  Ph.D.,  Floriculture. 

MORTIER  F.  BARRUS,  Ph.D.,  Plant  Pathology. 

CLYDE  H.  MYERS,  M.S.,  Ph.D.,  Plant  Breeding. 

GEORGE  W.  TAILBY,  JR.,  B.S.A.,  Superintendent  of  Livestock. 

EDWARD  S.  GUTHRIE,  M.S.  in  Agr.,  Ph.D.,  Dairy  Industry. 

JAMES  C.  BRADLEY,  Ph.D.,  Entomology. 

PAUL  WORK,  B.S.,  A.B.,  Vegetable  Gardening. 

JOHN  BENTLEY,  JR.,  B.S.,  M.F.,  Forestry. 

EARL  W.  BENJAMIN,  Ph.D.,  Poultry  Husbandry. 

EMMONS  W.  LELAND,  B.S.A.,  Soil  Technology. 

CHARLES  T.  GREGORY,  Ph.D.,  Plant  Pathology. 

WALTER  W.  FISK,  M.S.  in  Agr.,  Dairy  Industry. 

ARTHUR  L.  THOMPSON,  Ph.D.,  Farm  Management. 

ROBERT  MATHESON,  Ph.D.,  Entomology. 

MORTIMER  D.  LEONARD,  B.S.,  Entomology. 

FRANK  E.  RICE,  Ph.D.,  Agricultural  Chemistry. 

VERN  B.  STEWART,  Ph.D.,  Plant  Pathology. 

IVAN  C.  JAGGER,  M.S.  in  Agr.,  Plant  Pathology  (In  cooperation  with  Rochester  University). 

WILLIAM  I.  MYERS,  B.S.,  Farm  Management. 

LEW  E.  HARVEY,  B.S.,  Farm  Management. 

LEONARD  A.  MAYNARD,  A.B.,  Ph.D.,  Animal  Husbandry. 

LOUIS  M.  MASSEY.-A.B.,  Ph.D.,  Plant  Pathology. 

BRISTOW  ADAMS,  B.A.,  Editor. 

LELA  G.  GROSS,  Assistant  Editor. 

The  regular  bulletins  of  the  Station  are  sent  free  on  request  to  residents  of  New  York  State. 


CONTENTS 

PAGE 

Dissemination , 42 1 

Spore  germination 423 

Penetration 427 

Distribution  within  the  host  tissues 434 

Spore  formation  and  size 441 

A  similar  organism 442 

Bacteria  in  relation  to  Plasmodiophora  Brassicae 443 

Summary 448 

Bibliography 451 


5073-11 

419 


FIG.  95. 


DISEASED  CABBAGE  PLANT  SHOWING  THE  THIN  STALK  AND  THE  ABSENCE 
OF  A   HEAD 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS1 
CHARLES  CHUPP 

Such  an  extensive  literature  on  clubroot  of  cruciferous  plants  has  accu- 
mulated that  it  would  seem  impossible  for  any  one  point  to  have  escaped 
careful  consideration.  But  when  a  close  examination  is  made  of  all  the 
data,  it  soon  becomes  apparent  that  only  such  prominent  phases  as 
symptoms,  cytology  of  the  organism,  and  control  methods,  have  been 
dealt  with  extensively,  while  certain  other  less  conspicuous  features  have 
been  neglected.  There  still  remain  to  be  satisfactorily  solved  the  follow- 
ing problems:  (a)  the  part  played  by  swarm-spores  in  the  dissemination 
of  Plasmodiophora  Brassicae  Wor.,  the  organism  that  causes  clubroot; 
(b)  spore  germination;  (c)  the  manner  in  which  the  pathogene  enters  the 
host;  (d)  the  distribution  of  the  organism  thruout  the  tissues  of  the  root; 
(e)  formation  and  size  of  the  spores;  and  (f)  the  relation  of  bacteria  to 
the  normal  development  of  the  myxomycete.  It  is  for  the  solution  of 
these  problems  that  the  following  investigations  have  been  conducted. 

DISSEMINATION 

In  a  general  way  the  manner  in  which  the  spores  are  carried  is  known, 
altho  two  errors  are  often  met  with  in  popular  descriptions.  For  example, 
in  a  number  of  reports  (Atkinson,  1889,  Carruthers,  1893,  and  others)  2 
are  statements  implying  that  swarm-spores  swim  about  in  the  water 
of  the  soil  until  they  reach  a  cabbage  root.  In  a  way  this  is  correct, 
but  the  average  layman  at  once  pictures  the  swarm-spores  as  traveling 
from  row  to  row  of  plants  or  even  from  field  to  field.  Nothing  could 
be  more  erroneous,  for,  as  far  as  dissemination  is  concerned,  the  motility 
of  the  swarm-spore  plays  such  a  slight  part  that  it  need  not  be  considered. 
Its  energy  is  not  directed  in  a  straight  line,  and  the  very  minuteness  of  the 
organism  would  preclude  any  effective  locomotion  in  the  time  that  it 
remains  alive. 

In  order  to  test  the  distance  to  which  swarm-spores  may  travel  in  the 
soil,  a  box  two  feet  square  was  filled  with  clay  mixed  with  muck-  soil, 
and  diseased  roots  were  buried  in  one  end.  Cabbage  seeds  were  then  sown 
in  the  box,  care  being  taken  not  to  transfer  any  of  the  soil  from  the  place 
where  the  inoculum  was  inserted.  When  the  seedlings  over  the  area 

1  Also  presented  to  the  Faculty  of  the  Graduate  School  of  Cornell  University,  September,  1916,  as  a 
major  thesis  in  partial  fulfillment  of  the  requirements  for  the  degree  of  doctor  of  philosophy. 

ACKNOWLEDGMENT.  The  author  gratefully  acknowledges  the  helpful  suggestions  and  criticisms  offered 
him  by  Professor  H.  H.  Whetzel  and  others  in  the  Department  of  Plant  Pathology  at  Cornell  University. 

-  Dates  in  parenthesis  refer  to  bibliography,  page  451. 

42I 


422 


BULLETIN  387 


FlG.  96.      DISEASED   CABBAGE   SEEDLINGS 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS  423 

where  diseased  roots  were  buried  had  become  so  badly  infected  that  they 
began  to  wilt  and  turn  yellow,  all  the  plants  were  discarded  and  the  plat 
was  reseeded.  Different  crops  of  seedlings  were  thus  grown  for  almost 
a  year,  and,  altho  there  was  a  gradual  spread  of  the  organism,  it  was 
only  by  careless  watering  and  planting  that  the  pathogene  was  carried 
in  the  soil  to  all  parts  of  the  box. 

Cabbage  seeds  were  sown  in  a  greenhouse  plat  in  rows  ten  inches  apart, 
the  bottom  of  each  trench  being  first  lined  with  infested  soil.  Halfway 
between  these  rows  were  sown  other  rows,  in  the  trenches  of  which  no  in- 
fested soil  was  placed.  The  inoculated  plants  (fig.  96)  became  infected 
at  a  very  early  stage,  while  the  plants  that  were  only  five  inches  away 
from  the  spores  remained  healthy  until  they  were  almost  mature. 

A  few  authors  (Carruthers,  1893,  and  others)  claim  that  wind  is  an 
important  agent  in  spore  dissemination.  This  may  be  true  in  light, 
loose  soil,  and  in  localities  where  strong  winds  prevail,  but  in  none  of  the 
observations  made  by  the  writer  was  there  a  single  case  in  which  the 
presence  of  the  organism  could  be  explained  on  this  basis.  On  the  other 
hand,  many  of  the  fields  showed  that  if  the  soil  were  not  transferred 
by  some  agent  other  than  the  wind  the  pathogene  did  not  spread.  On 
Long  Island,  New  York,  a  certain  field  was  observed,  one  corner  of  which 
was  slightly  lower  than  the  adjoining  part.  This  corner  had  been  used 
for  a  garden  until  clubroot  became  so  prevalent  that  the  plat  was  no  longer 
profitable  for  the  raising  of  crucifers.  It  was  then  tilled  with  the  remainder 
of  the  field  for  three  years  while  various  crops  were  grown,  cabbages 
not  being  planted  again  until  the  fourth  year.  A  space  only  slightly 
larger  than  the  original  garden  then  displayed  the  presence  of  clubroot. 
If  wind  had  been  an  important  agent  it  would  have  had  an  opportunity 
here,  for  the  land  was  almost  level  and  the  soil  was  very  loose.  This 
was  only  one  of  several  cases  in  which  the  same  conditions  were  observed. 

SPORE    GERMINATION 

Very  few  persons  have  been  successful  in  germinating  the  spores  of 
Plasmodiophora  Brassicae,  and  of  those  few  who  have  been  so  fortunate, 
still  fewer  have  seen  the  actual  process.  Woronin  (1878)  gives  a  brief 
description  and  a  series  of  illustrations  which  have  been  copied  by  nearly 
all  later  writers  on  this  phase  of  the  subject.  The  general  experience, 
however,  seems  to  have  been  like  that  of  Maire  and  Tison  (1911)  while 
working  with  Tetramyxa  parasitica  Goebel.  They  saw  only  one  spore 
actually  germinating,  and  after  a  very  long,  tiresome  vigil  they  left  it 
for  a  few  minutes.  On  returning  from  their  temporary  absence  they  found 
that  the  phenomenon  had  been  completed.  Notwithstanding  these  diffi- 


424  BULLETIN'  387 

culties,  Eycleshymer  (1894)  not  only  found  swarm-spores,  but  also  found 
that  when  left  in  the  culture  for  a  few  days  these  apparently  fused 
into  larger  bodies,  thereby  reacting  in  much  the  same  manner  as  Kunkel 
(1915)  found  to  be  the  case  with  Spongospora  subterranea  (Wollr.) 
Johnson.  Kunkel  discovered  that  each  cell  of  a  spore  ball  produces  a 
single  uninucleate  amoeba  which  soon  fuses  with  others  of  its  kind  to 
form  a  small  plasmodium.  This  occurs  not  only  in  the  case  of  spores  in 
the  soil,  but  even  with  those  still  in  the  base  of  the  old  sorus. 

There  are  several  obstacles  to  be  encountered  in  trying  to  observe  the 
actual  emergence  of  the  protoplasmic  mass  from  the  old  spore  wall.  First, 
it  is  difficult  to  get  a  very  large  percentage  of  germination  unless  the  most 
favorable  conditions  are  present.  Secondly,  all  observations  must  be  made 
with  the  oil-immersion  objective.  When  the  protoplasm  is  about  half- 
way out,  the  spore  wall  and  the  emerging  protoplast  begin  to  move,  making 
it  hard  to  keep  them  in  focus  or  even  within  the  field.  Consequently, 
when  the  process  seems  almost  complete  there  is  a  sudden  swift  whirl, 
and  the  swarm-spore,  with  the  adhering  empty  wall,  darts  out  of  sight. 
When  located  again,  the  spore  wall  is  empty,  and  the  swarm-spore,  lost 
among  others,  is  impossible  of  identification.  For  this  reason  no  actual 
separation  of  the  protoplasm  from  the  spore  wall  has  been  seen,  but 
enough  of  the  process  has  been  observed  to  enable  investigators  to  deter- 
mine the  general  method  by  which  this  is  accomplished  and  to  be  sure 
that  a  spore  gives  rise  to  only  one  swarm-spore. 

It  was  soon  learned  that  spores  do  not  germinate  well,  if  at  a1!,  in  dis- 
tilled water,  and  further  that,  altho  from  one  to  five  per  cent  of  the  spores 
taken  directly  from  a  fresh  root  germinate  in  muck-soil  filtrate,  a  much 
larger  percentage  of  germination  can  be  obtained  by  exposing  the  roots 
to  freezing  temperatures  for  two  weeks  or  longer.  This  was  accomplished 
by  tying  the  roots  in  cheesecloth  and  burying  them  under  the  snow,  or 
in  summer  by  keeping  them  in  the  refrigerator  for  that  length  of  time. 
Drying  the  roots  also  seems  to  have  a  beneficial  effect  on  germination, 
altho  this  must  not  be  carried  to  the  extreme.  The  muck-soil  filtrate 
was  made  by  filling  an  ordinary  flowerpot  with  muck,  placing  it  over  a 
large  funnel  lined  with  filter  paper,  and  then  pouring  hot  water  on  the 
soil.  The  resulting  medium  was  of  an  amber  color  and  slightly  acid. 

Temperature  conditions  also  influence  germination  of  the  spores.  It 
was  practically  impossible  to  obtain  infection  in  the  greenhouse  during 
the  coldest  winter  months  when  the  temperature  was  from  10°  to  18°  C. 
The  spores  also  fail  to  germinate  at  ordinary  room  temperature  (from  1 6° 
to  21°  C.).  The  optimum  temperature  for  germination  proved  to  be  from 
27°  to  30°  C.  This,  however,  is  not  the  case  when  spores  are  placed  in 
test  tubes  on  agar  with  young  cabbage  seedlings,  for  under  such  conditions 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS  425 

infection  takes  place  at  a  temperature  of  from  16°  to  21°  C.  The  pres- 
ence of  the  host  seems  in  some  manner  to  exert  an  influence  which  to  a 
certain  extent  takes  the  place  of  that  offered  by  a  greater  amount  of  heat. 

Usually  the  first  sign  of  germination  is  a  swelling  of  the  spore,  which 
sometimes  becomes  a  third  larger.  This  occurs  within  a  period  of  from 
fifteen  minutes  to  eight  hours  after  the  spores  are  placed  in  the  medium, 
altho  the  best  time  for  examining  the  culture  proved  to  be  at  the  end  of 
six  hours.  After  the  swelling  of  the  spore  there  is  a  bulging  at  one  side. 
The  protoplasm  withdraws  from  near  the  opposite  wall  and  leaves  a 
nearly  hyaline  semicircle  about  two-thirds  of  the  distance  from  the  center. 
The  pressure  exerted  splits  the  wall  just  enough  to  permit  the  protoplasm 
to  ooze  out.  Unlike  Woronin  (1878)  and  Mangin  (1902),  the  writer  has 
never  observed  the  protoplasm  taking  the  various  shapes  that  these 
authors  assign  to  it,  but  while  oozing  out  it  collects  in  a  sphere  or  a  hemi- 
sphere against  the  wall  on  the  outside.  When  about  half  of  the  proto- 
plasm has  escaped,  the  whole  body  becomes  motile.  At  first  there  is  only 
a  trembling,  which  gradually  increases  in  violence  until  the  spore  is  turned 
around  entirely.  The  activity  now  becomes  so  great  that  it  is  with  diffi- 
culty that  the  microscope  is  kept  focused  on  it  correctly.  The  final  struggle 
is  apparently  a  rapid  spurt  across  the  field,  when  the  swarm-spore  is  lib- 
erated from  its  container,  and  at  once  begins  its  rotatory  activities.  The 
whole  process  under  the  microscope  consumes  an  hour  or  longer.  Evi- 
dently the  strong  light  turned  on  a  spore  retards  the  action,  for  in  many 
cases  the  spores  that  had  begun  to  germinate  when  placed  in  view  showed 
no  further  signs  of  development,  while  those  kept  in  the  dark  germinated 
much  more  rapidly  and  when  examined  at  the  end  of  the  same  period 
were  found  actively  swimming  about. 

A  considerable  part  of  the  contents  is  left  within  the  old  spore  wall, 
so  that  when  the  broken  part  is  turned  upward  it  has  the  appearance  of 
a  circle  bounded  by  a  darker  band,  the  width  of  which  is  about  one-third 
of  the  radius.  If,  however,  the  open  part  is  on  the  side,  the  residue  within 
the  spore  wall  resembles  more  nearly  a  crescent  (fig.  97). 

The  swarm-spore  when  alive  measures  from  1.7  to  3.5^  in  length, 
being  more  or  less  pyriform  with  a  thick  flagellum  at  the  smaller,  or 
anterior,  end  and  a  vacuole  near  the  posterior  end.  Unless  stained,  the 
flagellum  cannot  be  seen  under  the  microscope.  The  line  of  locomotion 
is  never  a  straight  one,  for  the  flagellum  is  lashed  about  by  the  beak, 
which  is  constantly  doubling  backward  so  that  a  whirling  motion  is  given 
to  the  swarm-spore.  Altho  the  latter  is  a  naked  mass  of  protoplasm, 
the  writer  has  never  seen  the  various  shapes  which  Woronin  (1878,  PL 
xxxiv)  has  pictured;  it  was  observed  in  every  case  to  be  globose  or  pyri- 
form, never  having  pseudopodia-like  structures. 


426 


BULLETIN  387 


It  has  been  difficult  to  properly  fix  swarm-spores  for  staining  flagella. 
The  first  method  of  staining  tried  was  that  ordinarily  employed  for  bac- 
teria, namely,  LoefHer's  mordant  and  Ziehl's  carbol  fuchsin.  When 
bacteria  were  in  the  mount  their  flagella  were  stained,  but  those  of  the 

swarm-spores  had  evi- 
dently disappeared.  The 
process  was  then  modi- 
fied slightly,  and  the 
cover-glass  mounts,  in- 
stead of  being  left  .to 
dry  in  the  incubator* 
were  placed  on  slides  in 
preparation  dishes  with 
ground-glass  tops.  In  the 
bottom  of  each  dish  was 
placed  a  few  cubic  centi- 
meters of  osmic  acid,  and 
the  lid  was  then  carefully 
fitted  in  place.  The  acid 
killed  a  few  of  the  swarm- 
spores  before  the  flagella 
could  be  withdrawn,  but 
never  a  very  large  pro- 
portion. Besides  demon- 
strating the  presence  of 
flagella,  the  stained  ma- 
terial also  displayed 
different  stages  of  ger- 
mination (fig.  97). 

Kunkel  (1915)  was  able 
to  get  spore  germination 
of  Spongospora  subter- 
ranea  on  an  agar  me- 
dium. Plasmodiophora 
Brassicae  evidently  does 
not  react  in  the  same 
way.  During  the  three 
years  of  the  present  work,  repeated  efforts  were  made  to  secure  not  only 
germination  on  the  surface  of  agar,  but  also  formation  of  plasmodia. 
Unless  the  spores  were  immersed  in  water  there  was  no  development. 
They  lay  there  until  the  agar  became  so  dry  that  they  finally  lost  their 
viability.  If  enough  of  the  muck-soil  filtrate  was  added,  the  swarm- 


FlG.    97.       SPORES    AND    SWARM-SPORES    OF    PLASMODI- 
OPHORA   BRASSICAE 

The  two  spores  at  the  top  have  already  germinated.  The  germi- 
nating spore  and  the  two  swarm-spores  near  the  bottom  were  drawn 
from  stained  mounts.  The  bacillus  shown  is  the  form  found  oftenest 
in  older  diseased  roots.  x  2100 


STUDIES  ox  CLUBROOT  _OF  CRUCIFEROUS  PLANTS  427 

spores  appeared  but  there  was  no  further  development.  They  were 
active  for  a  certain  time,  and  then  encysted  and  remained  in  that  con- 
dition as  long  as  the  cultures  were  kept.  This 
experiment  was  performed  on  four  kinds  of  agar 
media,  on  potato  plugs,  and  on  healthy  cabbage 
roots.  In  no  case  were  there  any  signs  of  further 
growth.  This,  with  subsequent  infection  experi- 
ments, indicates  very  strongly,  if  it  does  not  prove 
positively,  that  the  swarm-spores  never  fuse. 
This  is  in  keeping  with  what  has  been  found,  or 
>  at  least  suggested,  in  all  other  cases  of  parasitic 
slime  molds,  Spongospora  subterranea  excepted. 
If  spores  for  germination  are  taken  from  roots 

FIG.  98.   FLAGELLATE  OR- 

that  have  not  previously  been  disinfected,  there  GANISMS  ASSOCIATED 
are  often  found  in  the  cultures  flagellate  bodies  ™SICPALFASM°DIOPH°RA 
which  are  almost  small  enough  to  resemble 

swarmspores.  They  are  larger,  however,  are  more  active,  and  when  stained 
are  more  or  less  reniform,  having  two  flagella  arising  from  the  concave 
side  (fig.  98).  These,  as  pointed  out  later,  belong  to  another  organism. 

PENETRATION 

In  the  knowledge  of  the  life  history  of  Plasrnodiophora  Brassicae,  there 
has  always  been  a  gap  between  the  swarm-spore  stage  and  the  amoeba 
within  the  cell,  the  true  sequence  of  development  never  having  been 
shown.  Most  writers  pass  over  the  difficulty  with  the  mere  statement  that 
the  organism  enters  the  root  and  there  begins  its  parasitic  life.  Woronin 
(1878),  in  this  as  in  nearly  all  other  points  connected  with  clubroot,  is 
the  only  one  who  has  tried  to  fill  in  the  gap.  In  a  way  he  succeeded,  but, 
as  his  plants  died  before  reaching  the  stage  in  which  invasion  of  any  of 
the  tissue  took  place,  he  is  not  sure  that  the  root  hair  is  the  real  point  of 
entrance.  He  placed  cabbage  seedlings  in  shallow  watch  glasses,  in  water 
well  supplied  with  spores.  For  some  reason  the  plants  began  dying  before 
hypertrophy  took  place.  When  the  roots  were  examined  microscopically, 
the  root  hairs  were  filled  with  amoebae  but  nothing  further  had  happened. 
The  question  still  remained,  whether  these  infections  under  normal  con- 
ditions would  have  been  followed  later  by  invasion  of  the  cortical  cells, 
or  whether  the  case  was  similar  to  that  which  Schwartz  (1914)  found  in 
species  of  Ligniera.  Schwartz  thinks  that  penetration  takes  place  near 
the  apex  of  the  root,  so  that  when  the  root  hairs  act  as  bearers  of  the 
amoebae  the  parasite  does  not  advance  farther  than  the  base  of  the  cell. 

Most  writers  believe  not  only  that  the  apical  cells  and  the  root  hairs 
act  as  infection  courts,  but  also  that  the  epidermal  cells  can  be  infected 


428  BULLETIN  387 

directly  up  to  the  time  when  the  epidermal  layer  is  thrown  off  (Woronin, 
1878).  Somerville  (1895)  gives  an  observation  as  proof  of  this  statement. 
He  often  found  swellings  high  up  on  the  roots  of  turnips,  where  he  declares 
no  root  hairs  could  have  been  responsible  for  the  entrance  of  the  slime 
mold,  which  must  have  penetrated  the  thick  cuticle.  This  question  of 
entrance  has  a  direct  economic  bearing  on  control,  for,  if  Somerville 's 
statement  is  true,  Massee's  (1903)  assumption  is  certainly  erroneous. 
Massee  states  that  the  Cruciferae  can  be  attacked  only  during  seedling 
time,  and  that  after  six  weeks  they  are  practically  immune.  It  is  doubtful 
whether  either  Somerville  or  Massee  interprets  the  conditions  correctly. 
If  infection  could  not  take  place  after  six  weeks,  the  grower  could  control 
the  disease  merely  by  late  transplanting  and  the  proper  care  of  his  seed 
beds;  but  this  has  evidently  not  proved  to  be  the  case  in  practice. 

Maire  and  Tison  (1909,  1911)  and  Schwartz  (1910,  1911,  1914)  have 
done  nearly  all  the  work  that  has  been  reported  on  the  parasitic  slime 
molds  other  than  Spongospora  subterranea  and  Plasmodiophora  Brassicae. 
It  is  interesting  to  note  that  their  conclusions  agree  very  closely,  and  that 
they  feel  sure  the  amoebae  enter  oftener  thru  the  apical  cells  than  otherwise, 
altho  the  root  hairs  also  may  serve  as  points  of  entrance.  They  made  no 
particular  study  of  this  question,  but  were  led  to  this  conclusion  by  finding 
uninucleate  amoebae  in  the  cells  near  the  growing  tips.  Their  opinion  is 
substantiated  also  by  the  presence  of  rows  of  diseased  cortical  cells,  the 
divisions  of  which  apparently  take  place  when  still  very  near  the  initial 
cells  in  the  root  tips.  The  powdery  scab  pathogene,  Spongospora  sub- 
terranea, passes  directly  thru  and  between  the  epidermal  cells  into  the 
tuber  (Kunkel,  1915). 

There  is  more  or  less  difficulty  in  studying  the  nature  of  penetration 
in  the  case  of  Plasmodiophora  Brassicae,  because  of  the  fact  that  the 
uninucleate  amoebae  are  so  small.  They  can  be  recognized  only  under  a 
very  high  magnification,  and,  since  they  are  so  nearly  transparent,  stained 
sections  must  be  used  for  all  the  work.  A  very  large  number  of  both 
longitudinal  and  cross  sections  were  prepared,  the  thickness  ranging  from 
three  to  fifteen  microns,  and  the  staining  was  done  with  the  combination 
stains  of  safranin,  gentian  violet,  and  orange  G.  These  proved  best  for 
differentiating  the  parasite  from  the  host,  especially  when  orange  G  was 
used  in  excess. 

There  is  no  possible  stage  in  penetration  that  was  not  represented  in 
the  preparations.  Large,  as  well  as  very  small,  roots  were  sectioned, 
and  a  great  number  of  epidermal  cells  showed  amoebae.  But  in  a  careful 
study  of  almost  three  hundred  slides,  none  of  these  cells  showed  that 
penetration  had  taken  place  directly  thru  the  cutinized  wall.  In  a  number 
of  cases  this  appeared  to  be  true  when  the  sections  were  first  examined, 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS 


429 


but  a  more  detailed  study  of  the  same  series  showed  the  invaded  cell  to 
be  in  every  case  the  basal  portion  df  a  root  hair.  This,  together  with  the 
fact  that  no  new  swellings  are  ever  found  at  any  great  distance  from  the 
region  where  root  hairs  might  have  existed  previotisly,  has  led  the  writer 
to  believe  that  seldom,  if  ever,  is  there  direct  penetration  into  simple 
epidermal  cells. 

This  holds  true  not  only  for  the  area  above  the  place  where  the  root 
hairs  have  disappeared,  but  evidently  also  for  the  space  near  the  extreme 
tips  where  the  hairs  have  not  yet  been  formed.  Not  only  did  these  slides 
demonstrate  this  point :  but  infection  secured  under  aseptic  conditions 
in  test  tubes  has  confirmed  it.  The  small  root-tips  were  so  placed  that 
they  were  the  first  to  come  into  contact  with  particles  of  diseased  tissue 
and  the  muck-soil  filtrate  containing  free  spores.  When  these  rootlets 
were  sectioned  and  stained,  they  showed  various  stages  of  root-hair 
invasion,  but  no 
amcebas  were 
found  in  any  of 
the  apical  cells. 
The  evidence  pre- 
sented in  these 
slides  shows  that 
these  invasions 
are  not,  like  those 
which  Schwartz 
(1914)  suggested 
for  Ligniera  sp., 

confined  alone  to  the  epidermal  cells  of  which  the  hairs  are  outgrowths. 
The  passage  of  amoebae  from  the  epidermal  cells  into  the  cortical  tissue  is 
demonstrated  not  only  by  the  position  of  the  amcebas  within  the  paren- 
chyma cells,  but  also  by  actual  cell-wall  penetration. 

The  argument  advanced  for  other  species  of  Plasmodiophoraceae,  that 
infection  must  take  place  in  the  growing  tip  where  cells  are  dividing 
rapidly  because  the  organism  often  occurs  in  definite  rows  of  the  cortical 
cells,  does  not  necessarily  apply  to  Plasmodiophora  Brassicae.  A  glance 
at  a  section  of  a  root  tip  (fig.  99)  indicates  the  difficulty  that  a  swarm- 
spore  would  encounter  in  entering  at  this  point.  The  rootcap  does  not 
merely  protect  the  root  tip,  but  a  row  of  its  cells  extends  upward  almost 
halfway  to  the  root  hairs.  The  remaining  distance  is  protected  by  a 
comparatively  heavy  cuticle,  leaving  the  root  hair  as  practically  the  only 
vulnerable  point.  Moreover,  the  presence  of  the  organism  in  continuous 
rows  of  cells  can  be  explained  in  another  manner.  The  condition  shown 


FlG.   99.      LONGITUDINAL    SECTION   OF   A   CABBAGE   ROOT 

This  shows  the  tip  of  the  cabbage  root  protected  by  the  cells  of  the  root- 
cap.      X  no 


43° 


BULLETIN  387 


in  figure  IOO,B,  gives  no  indication  as  to  where  penetration  occurred. 
Yet  by  moving  the  section  the  length  of  half  a  dozen  cells,  there  is  seen 
an  uninterrupted  connection  of  diseased  tissue  between  this  particular 

row  and  the  epidermis 
(fig.  i oo, A). 

So  far  as  the  writer's 
observations  go,  there 
seems  to  be  no  question 
but  that  penetration 
does  take  place  thru  the 
root  hairs,  and  thru 
these  only.  Eycle- 
shymer  (1894)  suggests 
that  wounds  caused  by 
insects  may  provide  a 
means  of  entrance  for 
the  parasite.  This  is 
altogether  probable ;  yet 
the  writer  has  never 
observed  any  indica- 
tions of  this  condition, 
so  that  if  it  ever  hap- 
pens it  apparently  does 
so  very  rarely.  If  cul- 
tures could  be  secured 
within  pieces  of  healthy 
disinfected  roots  in  test 
tubes,  it  would  at  least 
be  evidence  that  such 
wound  infection  might 
take  place.  Pinoy(igo5) 
removed  small  pieces  of 
nealthy  roots  by  means 
of  sterilized  pipettes, 
and  by  inoculating  them 
TISSUE  OF  A  CABBAGE  with  spores  secured  cul- 
tures of  the  organism, 
provided  the  tubes  were 
sealed  so  that  the  aerobic 
bacteria  were  deprived  of  oxygen.  His  discussion  of  this  point  is  some- 
what lacking  in  clearness.  Besides,  the  time  in  which  he  claims  spores 
were  produced  in  the  roots  is  unusually  short.  He  gives  it  as  five  days, 


FlG.    IOO.      DISEASED 


CORTICAL 
ROOT 


A,  A  row  of  diseased  cortical  cells;  B,  another  row  of  diseased  cor- 
tical cells  connected  with  the  epidermis  by  an  unbroken  line  of  diseased 
tissue.  X  no 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS  431 

which  is  the  same  time  that  under  the  most  favorable  circumstances  it 
takes  swarm-spores  to  pass  thru  the  root  hairs  into  the  cortical  tissue 
and  to  develop  sufficient  hypertrophy  to  be  visible  to  the  naked  eye. 
Kleimenov  (1912)  tried  the  same  experiment  and  failed.  In  the  writer's 
experiments  it  was  also  tried  repeatedly,  always  with  failure.  If  the  cul- 
tures were  kept  free  from  bacteria  the  root  underwent  no  change.  If 
bacteria  were  added,  the  root  became  soft  and  foul-smelling,  whether  the 
test  tubes  were  closed  with  cotton  plugs  or  sealed  with  paraffin  over 
cork  or  cotton  stoppers.  Sealing  did  not  stop  the  growth  of  the  bacteria, 
as  Pinoy  claims  for  his  experiments. 

Altho  authors  popularly  describe  with  some  assurance  various  ways 
in  which  the  organism  may  enter  the  host,  no  one  has  observed  the  real 
process.  Even  Woronin,  who  believed  that  the  organism  passes  thru  the 
root  hair,  was  never  able  to  demonstrate  this  clearly.  Nevertheless  he 
felt  assured  that  it  enters  in  the  form  of  a  uninucleate  amoeba,  and  his 
ODinion  has  been  accepted  by  most  investigators.  A  few  workers,  such  as 
Worthington  G.  Smith  (1884),  maintain  that  the  organism  enters  the 
root  in  the  form  of  a  plasmodium,  but  this  theory  has  never  been  accepted 
generally.  The  question  was  revived  again  when  Kunkel  (1915)  studied 
the  powdery  scab  of  potato,  in  which  the  swarm-spores  are  found  to  fuse 
before  attacking  the  host. 

There  seems  to  be  no  doubt  in  the  minds  of  Maire  and  Tison  (1911) 
and  Schwartz  (1914)  that  all  the  other  known  parasitic  myxomycetes 
enter  immediately  after  the  swarm-spore  stage.  This  conclusion  is  based 
on  the  fact  that  many  of  the  slides  of  these  investigators  show  the  uninu- 
cleate forms  in  the  apical  cells.  There  is  no  other  theory  that  would  explain 
this  phenomenon,  unless  a  single  uninucleate  amoeba  of  an  infecting 
plasmodium  passes  thru  the  intervening  cell  walls  and  spreads  in  this 
manner  thru  the  tissue.  This  is  improbable. 

Because  of  the  diminutive  size  of  the  swarm-spore,  the  only  satisfactory 
method  for  studying  penetration  appears  to  be  by  means  of  stained  sec- 
tions of  roots  showing  the  earliest  stages  possible.  In  the  first  part  of  this 
work,  young  plants  from  the  greenhouse  were  used,  but  none  of  the  stages 
were  young  enough  to  give  just  what  was  desired.  An  attempt  was  then 
made  to  grow  plants  in  large  test  tubes  on  screens  so  arranged  that  the 
roots  were  hanging  in  muck-soil  filtrate  containing  a  heavy  suspension  of 
spores.  The  roots  did  not  develop  well  when  immersed  in  the  liquid 
medium,  and  but  few  root  hairs  were  present.  An  attempt  was  then  made 
to  grow  seedlings  in  soil,  in  flats  six  inches  square,  with  diseased  tissue  so 
plentiful  that  none  of  the  plants  could  escape  infection.  The  roots  were 
fixed  and  embedded  at  intervals  before  the  time  when  ordinary  symptoms 
became  apparent  to  the  naked  eye.  This  gave  nearly  all  the  early  stages 


432 


BULLETIN  387 


of  infection,  but  the  adhering  particles  of  soil,  which  could  not  be  washed 
off  without  sacrificing  the  hairs,  not  only  were  detrimental  to  the  micro- 
tome knife,  but  also  obstructed  a  clear  view  of  the  cell  walls.  Finally  a 
method  was  devised  whereby  infected  roots  could  be  procured  free  from 
any  other  contamination.  Diseased  roots  that  contained  spores  but  were 
not  far  enough  advanced  to  be  invaded  by  bacteria  were  sterilized  on  the 
surface  with  mercuric  chloride  and  transferred  to  agar  slants  in  test  tubes. 
After  two  weeks  cooling  in  the  ice  chest  they  were  finely  minced  in  the  agar, 

and  incubated 
until  it  was  clear 
that  no  bacteria 
were  present  in 
the  tissue,  from 
which  they  might 
have  been  liber- 
ated by  the  cut- 
ting. After  enough 
time  had  elapsed 
to  insure  perfect 
freedom  from  any 
saprophytes,  a  few 
drops  of  sterilized 
muck-soil  filtrate, 
and  a  young  cab- 
bage  seedling 
which  had  been 
grown  from  disin- 

FlG.    IOI.      THE    AMCEBA   OF   PLASMODIOPHORA    BRASSICAE   IN   A      f  ected  Seed  On  a^ar 
ROOT   HAIR  .          . 

A,  A   root   hair    with  an  amceba  showing  two  nuclei.     B,  A  uninucleate     ]  b    ' 

amceba  in  a  root  hair  which  shows  an  abnormal  swelling  in  the  immediate  were  added  It 
vicinity  of  the  organism.  C,  A  uninucleate  amoeba  in  a  tangential  section 

of  a  root  hair;  the  nucleolus  has  elongated,  as  it  ordinarily  does  just  before  WaS  neCCSSarV  to 
nuclear  division.  D,  A  host  nucleus  in  a  root  hair,  showing  its  size  as  com- 


pared  with  that  of  a  uninucleate  amoeba, 
shrunken,  distorted  root  hair.      X  1600 


E,  A  uninucleate   amoeba  in  a 


exercise     care    in 
adding     sufficient 

liquid  to  permit  spore  germination  and  not  have  an  excess,  which  would 
injure  the  root.  A  few  drops  would  not  evaporate  until  all  the  swarm- 
spores  had  ample  time  to  be  set  free  and  attack  the  root  hair.  The 
process  was  somewhat  long,  and  very  often  roots  were  chosen  which  were 
too  old  and  were  already  contaminated  with  bacteria.  In  spite  of  all  the 
difficulties,  enough  pure  cultures  were  obtained  to  provide  a  large  number 
of  sections  which  showed  all  sizes  of  amoebae. 

The  first  and  most  important  thing  shown  by  the  stained  sections  was 
that  Plasmodiophora  Brassicae  enters  the  root  hair  as  a  uninucleate  amceba, 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS  433 

not  as  a  plasmodium.  There  are  several  facts  that  prove  this  conclu- 
sively, even  tho  the  actual  phase  of  the  organism  passing  thru  the  wall 
was  never  observed  with  certainty.  A  number  of  slides  show  cases  that 
might  be  interpreted  as  actual  penetration,  but  as  the  nucleus  in 
no  case  appears  in  the  act  of  making  the  passage  one  cannot  be 
certain  of  such  an  interpretation.  Nevertheless,  numerous  cases  are 
to  be  found  of  a  uninucleate  amoeba  just  within  the  wall  of  the  root 
hair  and  far  enough  away  from  any  other  infection  to  preclude  all 
possibility  of  its  having  reached  there  except  by  entering  singly  thru  the 
wall  (fig.  101). 

Evidently  the  reason  why  no  one  has  recorded  this  stage  heretofore  is 
because  the  amoeba  hardly  enters  before  nuclear  division  and  growth 
takes  place.  Some  slides  show  binucleate  amoebae  still  within  the  hollow 
of  the  enlarged  cavity,  apparently  produced  by  the  stimulus  of  the  para- 
site. Other  sections  show  trinucleate  amoebae,  and  it  is  not  difficult  to 
find  amoebae  with  six  or  more  nuclei  (fig.  104,  page  436). 

This  series  of  stages  would  indicate  that  penetration  takes  place  in 
the  uninucleate  stage,  particularly  since  the  large  multinucleate  amoebae 
are  to  be  found,  in  nearly  every  instance,  near  the  base  of  the  root  hair, 
while  the  smaller  and  fewer-nucleate  amoebae  are  always  on  the  inside  of 
the  root-hair  wall  about  two-thirds  of  the  distance  from  the  base.  Amoebae 
are  seldom  found  in  the  tip  of  the  hair. 

Another  point  that  confirms  the  above  view  of  penetration  is  that  in 
the  absence  of  growing  host  roots  the  swarm-spores  develop  no  further 
when  the  spores  are  germinated  under  artificial  conditions,  and  after  a 
short  period  of  activity  the  swarm-spores  encyst  and  eventually  die. 
If  plasmodia  are  formed  under  normal  conditions,  there  should  have  been 
at  least  a  suggestion  of  this  in  a  few  of  the  numerous  cultures  used  in 
the  experiments. 

In  this  connection  also  the  very  interesting  question  of  sexual  fusion 
arises.  It  is  believed  by  several  cytologists  that  there  are  two  nuclear 
divisions  just  before  spore  formation  and  that  one  of  these  is  probably  a 
reduction  division.  If  this  is  true,  it  would  imply  that  somewhere  in  the 
life  cycle  there  has  been  a  fusion.  Winge  (1913)  and  others  believe  that 
this  occurs  among  the  swarm-spores  before  they  enter  the  host.  Prowazek 
(1905)  is  of  the  opinion  that  the  amoebae  within  the  host  unite  and  then 
the  nuclei  fuse.  Even  Nawaschin  (1899)  believes  this  union  takes  place, 
but  apparently  he  thinks  it  is  of  no  significance  in  reproduction.  Maire 
and  Tison  (1909,  1911)  have  disproved  the  amoebal  union,  and  their  view 
is  certainly  correct,  for  it  is  possible  to  find  slides  showing  one  amoeba 
breaking  up  into  spores  while  in  another,  immediately  adjoining,  division 


434 


BULLETIN  387 


has  not  yet  begun  (fig.  102,  D).  On  the  other  hand,  it  would  seem  that  the 
fusion  of  two  swarm-spores  would  give  an  increase  in  size,  but  the  measure- 
ments of  amoebae  just  after  penetration  show  them  to  be  no  larger  than 
the  swarm-spores  just  out  of  the  spore  wall.  Consequently  Winge's  theory 


FlG.    102.       SPOKES    AND    AMCEB^E    OF    FLASMODIOPHORA    BRASSICAE 

A,  Spores  before  their  final  separation  from  one  another;  B,  cell  filled  with  amoebae;  C,  cell  filled  with 
spores.  All  X  800.  D,  Formation  of  spores,  X  Soo 

must  be  discarded.     It  thus  appears  that  the  real  fusion  stage,  if  there  is 
one,  is  still  to  be  discovered. 

DISTRIBUTION    WITHIN    THE    HOST    TISSUES 

As  stated  above,  the  uninucleate  amoeba,  just  after  its  entrance  into  the 
host,  lies  at  first  in  a  small  cavity  produced  by  the  outward  swelling  of 
the  part  of  the  root  hair  at  the  point  where  the  organism  entered.  This 
protuberance  is  no  doubt  caused  by  the  irritating  presence  of  the  para- 
site (fig.  10 1,  A,  B,  E).  Following  penetration  the  amoeba  increases  in  size 
and  pushes  toward  the  center  of  the  hair.  The  movement  is  accomplished 
by  an  actual  amoeboid  creeping,  and  an  elongation  and  gradual  segmen- 
tation of  the  forward  part.  Woronin  (1878)  was  able  to  observe  the 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS 


435 


former  method  of  locomotion  in  the  living  cells,  and  mentions  it  as  the 
means  by  which  the  organism  moves.  Schwartz  (1910),  on  the  other 
hand,  observed  the  growing  of  the  amoeboid  tip  in  Ligniera  Jumi  (Sch.) 
M.  et  T.,  and  explains  the  change  of  position  on  that  basis  alone.  A  root 
hair  is  shown  in  figure  104,  D,  which  apparently  was  infected  near  the  tip, 
and  as  the  organism  grew  rootward  fission  took  place,  so  that  when  the 
anterior  part  of  the  amoeba  eventually  reached  the  base  of  the  cell  the 
root  hair  was  filled  completely  with  the  meronts,  as  Maire  and  Tison 
(1911)  designate  the  segmented  parts  (fig.  103).  This  does  not  always  take 
place,  for  there  were  many  more  cases  observed  in  which  the  intact 
amoeba  reached  the  base  of  the  cell  (fig.  104,  E,  F).  In  either  case,  if  the 
time  consumed  is  too  long,  or  if 
for  any  other  reason  sporulation 
begins,  the  amoeba  loses  its 
power  of  further  penetration 
into  the  cortical  tissues.  If, 
however,  it  reaches  the  inner 
wall  of  the  root-hair  cell,  its 
pseudopodia  are  extended  into 
the  very  smallest  thread-like 
processes,  which  pass  thru  and 
into  the  cortical  cell  (fig.  105, 
E,  F,G).  Schwartz (1910), in  de- 
scribing penetration  by  Ligniera 
Junci,  gives  the  same  route  of 
invasion  but  does  not  state  how 
the  passage  from  the  epidermis 
into  the  cortical  cells  takes  F  '"  l-°3'  PHOTOMICROGRAPH^  CELLS  CONTAIN- 

place.       ThlS    question    IS    OI    eS-     One  amoeba  has  elongated  considerably  and  is  separating 

pecial    interest,    since    in    the 

latter  part  of  his  discussion  Schwartz  states  his  belief  that  amoebae  never 
have  the  power  of  penetrating  cell  walls.  There  is  no  other  apparent 
means  by  which  this  could  be  accomplished,  for  the  epidermal  cells  seldom 
divide  periclinally. 

It  would  be  difficult  to  explain  the  wide  distribution  of  the  parasite 
within  the  root  if  cell-wall  penetration  did  not  occur,  even  tho  it  were 
taken  for  granted  that  invasion  begins  in  the  apical  cells.  The  rootcap 
s •)  fully  protects  these  rapidly  dividing  primary  cells  that  one  must  pre- 
suppose that  in  order  to  reach  them  the  organism  can  pierce  the  walls. 
Then,  in  the  maturer  roots  constant  secondary  thickening  by  the  cam- 
bium takes  place,  which  would  ultimately  push  most  of  the  diseased  cells 
toward  the  periphery  or  isolate  them  near  the  center.  This,  however,  does 


436 


BULLETIN  387 


FlG.    IO4.       SECTIONS    OF    CABBAGE    ROOT    HAIRS    SHOWING    AMOEBAE 

A,  An  amoeba  dividing  by  fission  in  a  root  hair.  B,  A  much  distorted  and  swollen  root  hair,  with 
a  small  amoeba  partly  surrounding  its  nucleus,  which  is  also  much  enlarged.  C,  An  amoeba  near  the  tip 
of  a  root  hair;  the  nucleoli  are  elongated,  as  they  ordinarily  are  just  before  nuclear  division.  D,  A  root  hair 
filled  with  meronts.  E  and  F,  Amoebae  in  epidermal  cells  of  the  root  and  at  the  base  of  root  hairs;  amcebae 
about  to  break  up  into  spores.  G,  A  root  hair  filled  with  an  amoeba.  H,  A  root  hair  filled  with  amoebae 
breaking  up  into  spores;  the  vacuolar  channels  between  each  nucleus  are  plainly  visible.  X  600 


STUDIES  ox  CLUBROOT  OF  CRUCIFEROUS  PLANTS 


437 


PlG.    105.      AM(EB,E   IN    THE    HOST    CELLS 

A,.  B,  C,  D,  and  G,  Amoebae,  with  pseudopodia,  in  recently  infected  roots.  E,  Amcebae  in  adjoining 
cells,  divided  only  by  the  cell  walls;  this  is  evidently  a  case  in  which  penetration  occurred,  altho  no  con- 
necting strands  are  visible.  F,  Amoeba  penetrating  the  cell  wall.  X  no 


BULLETIN  387 


not  happen,  as  may  be  seen  by  examination 
of  cross-sections.  Besides,  if  it  did,  it 
would  explain  only  the  presence  of  longi- 
tudinal rows  of  diseased  cells,  and  not  nec- 
essarily the  whole  "  Krankheitsherde."  For 
example,  in  figure  106  the  original  cortex 
was  five  cells  wide.  That  is  the  same  number 
as  is  found  in  the  "  Krankheitsherde." 
These  are  connected  by  a  single  row  of 
diseased  cells.  How  could  the  diseased 
area  have  originated  without  direct  migra- 
tion and  still  show  no  radial  hyperplasia? 

Woronin's  (1878)  view  is  that  the  para- 
site, taking  advantage  of  the  pits  found 
in  the  parenchyma,  goes  directly  from  cell 
to  cell  and  thus  thruout  the  root,  much 
like  Spongospora  subterranea  in  tubers  as 
described  by  Kunkel  (1915)  except  that  the 
organism  in  the  potato  is  intercellular.  To 
Nawaschin  (1899),  who  saw  no  actual  pas- 
sage thru  the  walls,  it  seemed  too  difficult 
a  task  for  the  amoeba  to  break  thru  the 
plasma  membrane;  hence  he  decided  that 
there  is  never  any  migration,  the  distri- 
bution being  due  entirely  to  rapid  division 
of  diseased  cells. 

Maire  and  Tison  (1909,  1911)  and 
Schwartz  (1910,  1911,  1914),  who  have 
made  observations  on  the  other  Plasmo- 
diophoraceas,  explain  the  scattered  diseased 
areas  as  due  to  infection  of  the  apical  cells 
which  by  subsequent  divisions  gives  rise  to 
the  diseased  rows  so  often  seen.  Schwartz 
(1911),  in  spite  of  the  fact  that  he  saw 
pseudopodia  in  Sorosphaera  graminis  ex- 
tending thru  the  cell  wall,  makes  the  state- 
ment that  he  does  not  believe  species  of  any 

FIG.  106.  FORMATION  OF  "  KRANK-  of  the  genera  show  direct  migration.     He 
HEITSHERDE"  explains    his    skepticism    on    the    ground 

im^^d-^SeySJS'S^ed^aS  that    he    never    saw    any    accompanying 

in  number  thru  outward  pressure  of  the  -  .        ,  <  <• 

hypertrophied  ceils,    x  no  nucleus  in  these  pseudopodia. 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS 


439 


Lutman  (1913)  figures  actual  passage  thru  the  wall.  He  believes  that 
the  amoebae  are  transferred  in  the  cortical  tissue  both  by  penetration  and 
by  division  of  the  host  cells. 

It  is  altogether  possible  to  cut  a  large  number  of  sections  without  obtain- 
ing any  definite  clue  as  to  the  mode  of  migration  from  the  root  hair  to  the 
cortex  or  the  medullary  ray,  for  in  the  later  stages  the  cell  wall  acts  as  a 
perfect  barrier.  In  view  of  this  fact,  Nawaschin  might  have  done  enough 
staining  to  complete  his  carefully  planned  cytological  problem  without 
once  cutting  a  root  so  recently  infected  that  the  passage  from  one  cell  to 
another  could  be  detected.  During  the  first  two  years  of  the  writer's 
study,  only  roots  that  showed  evident  hypertrophy  were  used  and  none 
of  these  gave  any  evidence  of  such  a  passage.  As  soon  as  the  smallest 
rootlets  were  sectioned  longi- 
tudinally, penetration  could  be 
observed.  It  is  true  that  it  never 
appeared  abundantly;  yet  it 
might  have  been  there  and  not 
noticed,  for  the  opening  in  the 
wall  is  so  minute  and  the  strand 
which  passes  thru  is  so  nearly 
hyaline  that  only  deep  staining 
will  make  it  apparent  under  the 
microscope  (fig.  105,  F,  page  437). 
There  are  numerous  cases  in 
which  it  is  probable  that  such  a 
migration  has  taken  place  but 
the  connecting  strand  cannot  be 
seen  (fig.  105,  E). 

The  objection  has  been  sug- 
gested that  these  strands  are  merely  the  remains  of  a  thread  which 
was  not  severed  when  the  wall  was  laid  down  between  two  dividing  cells. 
This  may  be  true  in  such  cases  as  are  represented  in  figure  107,  but  in 
other  cases  the  position  of  the  cells  precludes  the  tenability  of  such  an 
assumption. 

Another  argument  in  favor  of  cell-wall  penetration  is  the  shape  of  the 
amoeba  in  the  initial  stages  of  invasion  as  compared  with  that  in  later 
stages.  When  the  smallest  rootlets,  containing  only  a  few  diseased  cells, 
are  sectioned  longitudinally,  the  amoebae  are  usually  seen  to  be  elongated 
and  often  have  pseudopodia  extending  in  different  directions.  This  is 
never  true  in  a  more  advanced  stage.  The  amoebae  are  then  nearly 
spherical  and  remain  stationary  in  the  cell.  This  difference  is  seen  on 
comparison  of  figures  105  and  108. 


FlG.   IO7.     AMCEBA  EXTENDING  FROM  ONE  CELL 
INTO    ANOTHER 


440 


BULLETIN  387 


The  small  offspring  at  once  begins  to  grow  in  the  newly  invaded  cell, 
the  process  of  penetration  being  repeated  while  the  tissue  is  still  young. 
From  this  statement,  however,  it  is  not  to  be  inferred  that  all  this  occurs 
while  there  is  no  cell  division,  and  that  each  daughter  cell  in  turn  does 
not  become  infected.  Cell  division  certainly  does  take  place  from  the 
beginning,  first  in  conjunction  with  penetration  and  later  alone.  The 
result  of  both  methods  of  invasion  is  illustrated  by  figure  100,  B  (page  430), 
which  shows  a  row  of  eight  diseased  cells.  They  extend  the  same  length  as 
three  healthy  cells.  Their  relative  lengths  had  been  attained  before 
infection  occurred;  therefore  the  organism  must  have  passed  thru  at 
least  two  walls,  while  cell  division  accounts  for  the  remainder. 

This  leads  the  study  to  the 
process  of  "  Krankheitsherde " 
formation.  The  whole  subject 
has  usually  been  dismissed  with 
the  arbitrary  statement  that  a 
single  cell  becomes  diseased  and 
then  a  closely  packed  group  of 
cells  finally  results  by  repeated 
divisions  both  anticlinally  and 
periclinally.  A  cursory  study  of 
cross  sections  would  naturally 
suggest  such  an  explanation,  for 
undoubtedly  the  diseased  areas 
are  arranged  in  more  or  less 
distinct  groups.  But  again  lon- 
gitudinal sections  of  young  and 
recently  infected  rootlets  may 
be  used  to  clear  up  the  difficulty 
and  show  the  initial  stages  of  a  typical  "Krankheitsherde." 

The  impression  must  be  avoided  that  passage  thru  cell  walls  is  so  fre- 
quent that  a  single  root-hair  infection  will  suffice  to  spread  the  organism 
thruout  the  entire  affected  part  of  the  root.  There  must  be  repeated 
infections,  since  the  amoebae  never  migrate  far,  as  the  longitudinal  sec- 
tions show.  They  may  enter  in  almost  a  straight  path  as  far  as  the  endo- 
dermis.  The  invaded  cells  may  then  divide  or  merely  increase  in  size. 
Meanwhile  the  adjoining  healthy  cells  show  abnormal  division.  Nawaschin 
(1899)  explains  this  hyperplasia  on  the  part  of  non-invaded  cells  as  due 
to  the  mechanical  outward  pressure  of  the  much-enlarged  diseased  cells. 
Eleven  rows  of  uninvaded  cells  adjacent  to  a  "Krankheitsherde"  are 
shown  in  figure  106  (page  438);  in  the  healthy  part  of  the  root  there  are 
only  five  rows  of  cells. 


FIG.  1 08. 


HOST  CELLS  FILLED  WITH  CLOSELY 
CROWDED    AMCEB^E 


STUDIES  ON  CLUHKOOT  OF  CRUCIFEROUS  PLANTS  441 

For  some  unknown  reason  the  amoeba  in  some  cases  may  not  penetrate 
as  far  as  the  endodermis,  but,  after  having  reached  a  certain  row  of  the 
cortical  cells,  it  may  pass  upward  or  downward  in  that  row,  cell  division 
taking  place  as  fast  as  invasion  occurs.  This  produces  such  rows  of  cells 
as  are  illustrated  in  figure  100.  The  vertical  direction  may  be  changed  at 
any  time  and  at  frequent  intervals  to  a  horizontal  one,  and  the  adjacent 
rows  thus  affected  at  once  begin  cell  division  in  each  direction.  The  result 
is  a  true  (<  Krankheitsherde."  Any  one  of  these  diseased  rows,  or  several 
of  them,  may  extend  beyond  another  group  of  healthy  cells,  from  which 
the  organism  again  moves  horizontally.  This  will  cause  a  second  "  Krank- 
heitsherde," above  or  below  the  first  and  separated  by  the  length  of  one 
or  more  healthy  cells.  This  is  illustrated  in  figure  106  better  than  it  can 
be  described.  A  single  infection  may  in  this  manner  give  rise  to  from 
one  to  probably  six  "Krankheitsherde,"  with  the  intervening  uninvaded 
cells  much  increased  in  number  over  those  in  the  normal  tissue. 

It  has  already  been  pointed  out  why  this  longitudinal  movement  is 
interpreted  as  the  result  of  cell- wall  penetration  instead  of  as  being  due 
to  mere  division.  The  diseased  area  shown  in  figure  106  is  only  five  rows 
of  cells  in  width.  The  perfectly  normal  tissue  of  the  same  root  shows 
exactly  the  same  number  of  rows.  There  has  been  no  periclinal  division, 
and  therefore  direct  migration  must  have  taken  place. 

All  this  occurs  when  the  root  is  only  a  few  millimeters  in  diameter. 
For  some  reason  the  walls  finally  become  impenetrable,  and  the  amoebae 
become  more  nearly  globose  and  later  are  transformed  into  masses  of 
spores. 

SPORE    FORMATION    AND    SIZE 

For  the  purpose  of  this  discussion  the  nuclear  phenomena  need  not 
be  included.  The  generally  accepted  explanation  of  a  true  mitotic  division 
followed  by  vacuolar  separation  into  individual  uninucleate  spores  is  well 
known.  This  separation  is  supposed  to  take  place  almost  simultaneously, 
but  stained  sections  do  not  always  show  this  to  be  true.  Stages  from  the 
amoeba  to  the  mature  spore  are  represented  in  figure  102,  D  (page  434). 
In  this  case  there  were  repeated  successive  separations  instead  of  simul- 
taneous fission,  so  that  each  amoeba  is  divided  into  two,  theji  four,  and  so 
on  until  no  two  nuclei  longer  remain  together.  The  unstained  spores  in 
figure  1 02,  A,  show  the  same  method  of  formation.  They  may  then  be 
hexagonal  or  irregular  in  outline,  and  much  larger  than  when  mature, 
but  they  soon  become  spherical. 

It  was  surprising  to  note  the  wide  difference  between  the  actual  size 
of  the  spores,  and  the  measurements  (i.6/z)  given  by  Woronin  (1878) 
and  nearly  all  succeeding  authors.  Molliard  (1909)  gives  the  diameter 


442 


BULLETIN  387 


of  the  spores  as  from  1.8  to  2.211,  and  Pinoy  (1907),  altho  he  does  not 
state  directly,  says  in  speaking  of  swarm-spores  that  they  are  from  3  to  4  n 
in  diameter. 

The  measurements  made  in  connection  with  the  present  experiments 
agree  more  nearly  with  those  of  Pinoy  for  the  swarm-spores.  The  spores 
in  formation,  when  not  yet  spherical,  measure  from  2.5  to  6.9  p.  in  diameter, 
being  much  more  variable  than  those  that  are  older.  The  smallest  mature 
spore  measured  was  1.9  j*,  and  the  largest  was  4.3  JJL.  These  measurements 
include  not  only  living  spores  but  also  those  stained  in  various  ways. 
The  average  was  3.3  //. 

A    SIMILAR    ORGANISM 

For  some  time  the  writer  was  at  a  loss  for  an  explanation  of  the  occasional 
presence  of  from  two  to  twelve  strange  nuclei  in  certain  root  hairs  and 
epidermal  cells  (fig.  109,  A).  These  are  from  3  to  4  n  in  diameter, 


B 

FlG.    109.       AN  UNKNOWN  ORGANISM  ASSOCIATED  WITH  PLASMODIOPHORA  BRASSICAE 

A,  Nuclear-like  bodies  in  a  root  hair,  probably  swarm-spores  of  Olpidium  Brassicae;  B  and  C,  an 
unknown  organism  in  the  epidermal  cells  of  a  cabbage  root,  probably  Olpidium  Brassicae.      X  800 

being  smaller  than  the  nuclei  of  the  host  cells.  The  nucleoli  have  a 
much  denser  content  than  those  of  the  host  cells,  and  are  much  smaller 
and  less  prominent.  They  appear  to  be  entire  swarm-spores  containing 
no  visible  cytoplasm;  however,  they  do  not  resemble  those  of  Plasmodi- 
ophora  Brassicae,  being  larger,  and,  most  important  of  all,  not  having  the 
hyaline  zone  about  the  nucleolus  which  is  so  characteristic  of  the  latter. 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS  443 

Furthermore,  amxba-like  bodies  are  found  in  the  epidermal  cells  or  the 
layer  next  to  them,  which  also  look  very  much  like  Plasmodiophora 
but  seem  to  be  inclosed  in  a  delicate  wall.  Stages  have  been  found  in  which 
each  of  these  bodies  has  an  appendage,  or  neck,  which  protrudes  thru 
the  outer  epidermal  cell  wall  (fig.  109,  B,  c).  The  organism  compares  very 
closely  with  that  described  by  Woronin  (1878)  as  Synchytrium  Brassicae 
and  later  by  Dangeard  (1886)  as  Olpidium  Brassicae.  Positive  proof  of 
its  identity  is  lacking. 

The  chytrid  never  produces  any  hypertrophy  or  other  outside  symptoms 
by  which  a  diseased  plant  can  be  recognized,  so  that  specimens  were 
found  only  accidentally.  For  this  reason  it  was  impossible  to  study 
the  swarm-spore  stage  or  the  details  of  the  life  history  of  the  organism. 

The  organism  evidently  enters  by  way  of  the  root  hairs,  and  never 
penetrates  far  into  the  host.  None  of  the  invaded  cells  are  changed  in 
size  or  general  appearance.  Even  the  invaded  root  hair  does  not  have 
that  slight  enlargement  which  has  been  mentioned  in  connection  with 
the  entrance  of  the  uninucleate  amoeba  of  Plasmodiophora  Brassicae. 

The  fungus  can  infect  a  plant  that  seems  perfectly  healthy.  'At  least 
it  is  not  a  saprophytic  form  following  the  clubroot  organism,  as  roots 
were  sectioned  which  showed  it  alone. 

BACTERIA    IN    RELATION    TO    PLASMODIOPHORA    BRASSICAE 

Pinoy's  (1902,  1903,  1905,  1907)  work  with  Myxomycetes  in  their 
relation  to  bacteria,  and  his  subsequent  suggestion  that  there  is  a  true 
symbiosis  between  the  two,  represent  a  very  interesting  phase  in  the  study 
of  Plasmodiophora.  It  has  long  been  held  that  certain  saprophytic  slime 
molds  feed  on  accompanying  organisms,  and  the  data  at  hand  seem  entirely 
plausible.  Lister  (1894)  has  seen  the  ingestion  of  bacteria  by  active 
swarm-spores.  The  experiments  of  Vuillemin  (1903),  Nadson  (1901), 
and  Potts  (1902)  show  that  Dictyostelium  mucoroides  Bref.  feeds  directly 
on  bacterial  colonies  and  destroys  a  large  number  of  these  at  fruiting 
time. 

The  general  conditions  of  subsistence  governing  saprophytic  forms 
and  those  controlling  parasitic  organisms  are  not  the  same,  however; 
so  that  from  a  priori  reasoning  it  would  seem  justifiable  to  say  that  Plas- 
,;iodiophora  Brassicae  needs  no  concomitant  organism  in  its  life  cycle. 
Yet  the  case  is  not  so  clear,  since,  on  examination  of  nearly  every  root 
that  has  been  diseased  for  some  time,  such  an  organism  is  found  to  be 
present.  When  the  surface  of  these  roots  is  sterilized  and  placed  in  agar, 
they  may  show  no  indication  of  bacteria  until  they  are  cut  in  two  and 
the  fresh  surface  is  placed  in  contact  with  the  medium.  Moreover,  E.  F. 


444  BULLETIN  387 

Smith  (1911)  and  Eycleshymer  (1894),  both  careful  workers,  state  that 
they  saw  these  bacteria.  This  is  also  in  accordance  with  what  Maire  and 
Tison  (1911)  claim  to  be  true  for  certain  parasitic  slime  molds  that  are 
able  to  ingest  unicellular  algas  present  in  their  aquatic  host;  and  with 
what  Kunkel  (1915)  has  demonstrated  in  the  case  of  Spongospora  sub- 
terranea  grown  on  agar  in  which  pure  cultures* of  plasmodia  become 
abnormal  and  die,  while  those  with  which  bacteria  are  present  live  and 
thrive. 

All  of  Pinoy's  (1905)  experiments  appear  to  corroborate  his  idea  that 
a  coccus  form  enters  the  root  with  the  swarm-spore  and  lives  in  constant 
association  with  the  parasite  thruout  its  entire  life  cycle.  He  stained 
sections  of  the  root  and  observed  bacterial  forms  within  the  cells.  They 
appeared  so  much  like  parts  of  the  cell  contents  that  the  microscopical 
analysis  had  to  be  accompanied  by  cultural  study.  For  this  he  procured 
diseased  roots  of  Brassica  sinensis  measuring  from  eight  to  ten  centi- 
meters in  diameter,  seared  the  outside,  and  cut  plugs  from  the  interior 
by  means  of  a  flamed  pipette.  When  these  plugs  were  planted  in  agar 
media,  numerous  colonies  of  bacteria  soon  appeared.  To  prove  that 
these  organisms  were  necessary  for  the  development  of  the  myxomycete, 
Pinoy  placed  spores  of  Plasmodiophora  Brassicae  in  a  large  number  of  test 
tubes  containing  sterilized  extract  of  roots.  In  two  tubes  the  spores  were 
accidentally  not  associated  with  bacteria  and  they  failed  to  germinate, 
while  in  all  the  other  tubes  the  spores  did  germinate. 

Pinoy's  results  are  interesting,  altho  the  work  does  not  appear  to  be 
extensive  enough  to  warrant  the  conclusion  he  has  drawn.  The  following 
experiments  were  undertaken  by  the  writer  in  further  quest  for  facts  bearing 
on  this  problem : 

Thruout  three  years  of  study  almost  five  hundred  petri-dish  and  test- 
tube  cultures  have  been  made  from  diseased  roots  of  all  sizes  and  ages, 
grown  under  various  conditions  and  in  widely  separated  localities.  The 
ordinary  method  of  procedure  was  to  place  the  roots  for  ten  minutes  in 
mercuric  chloride  i-iooo;  then,  after  rinsing  them  several  times  in  sterilized 
distilled  water,  to  break  the  roots  open  and  remove  bits  of  the  diseased 
tissue  from  the  broken  surface  by  means  of  a  flamed  scalpel.  The  bits 
of  roots  were  placed  in  a  sterilized  petri  dish,  where  they  were  teased  apart 
in  a  few  drops  of  sterilized  distilled  water.  Two  successive  dilutions 
were  made,  and  these,  together  with  the  original  drop  in  which  the  tissue 
had  been  crushed,  were  poured  into  nutrient  agar  media. 

All  the  results  were  uniform  in  that  no  bacterial  colonies  were  obtained 
from  the  roots  with  young  swellings.  From  the  medium-sized  swellings 
occasional  colonies  developed;  and  from  the  larger  galls,  especially  when 
the  epidermis  had  been  broken,  numerous  colonies  always  appeared. 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS  445 

The  fact  that  only  the  small  swellings  show  no  contamination  might 
be  attributed  to  the  penetration  of  the  mercuric  chloride.  In  order  to 
avoid  this  source  of  error,  the  time  of  treatment  was  reduced  from  ten 
minutes  to  five,  and  even  to  three,  or  was  dispensed  with  entirely,  the  roots 
being  soaked  for  three  hours  in  water  that  had  been  standing  over  calcium 
hypochlorite  for  two  hours  and  then  decanted.  The  results  were  the  same. 

Another  possible  hindrance  to  the  appearance  of  colonies  at  first  might 
have  been  the  medium,  which  was  nutrient  agar.  In  order  to  eliminate 
this  objection,  later  cultures  were  made  both  in  potato  agar  and  in  a 
medium  made  from  the  extract  of  healthy  cabbage  roots  like  that  used 
successfully  by  Kleimenov  (1912).  No  bacterial  growths  were  obtained. 

Bacteria  have  been  found  in  large  roots  similar  to  those  that  Pinoy 
used;  but  Pinoy  obtained  a  coccus,  while  the  most  prevalent  form  in 
the  cultures  of  the  writer  has  been  a  very  motile  rod-shaped  bacterium 
producing  yellowish,  opalescent  colonies  on  the  various  media.  In  test 
tubes  containing  disinfected  diseased  roots  this  organism  readily  produces 
a  soft  rot  and  thus  liberates  the  spores  of  the  slime  mold.  It  is  well 
known  that  the  epidermis  is  soon  ruptured  after  swelling  begins,  and  from 
all  indications  the  conditions  are  propitious  for  the  entrance  of  any 
T  organisms  that  may  be  in  the  soil.  This  is  doubly  true  for  any  that 
find  exposed  cabbage  tissue  a  favorable  substratum  on  which  to  reproduce, 
as  does  evidently  the  bacterium  mentioned  above.  These  series  of  cultures 
tend  to  show  that  bacteria  do  not  enter  with  the  swarm-spore,  as  Pinoy 
(1905)  believes,  but  that  the  disease  must  advance  to  a  certain  stage 
before  the  bacteria  can  gain  entrance.  The  above  experiments  are 
perhaps  in  themselves  not  sufficient  proof,  especially  since  they  bear 
on  the  negative  side  of  the  question.  To  these,  however,  are  to  be  added 
the  following  data: 

The  writer  has  found  that  spores  germinate  better  if  they  have  been 
exposed  to  cold  or  to  drying  for  a  short  time  before  being  placed  in  a 
warm  oven  at  a  temperature  of  from  2  7°  to  30°  C. ;  and  that  the  best  medium 
tested  is  water  that  has  been  filtered  thru  muck  soil.  Accordingly  diseased 
roots  were  washed,  treated  with  either  mercuric  chloride  or  calcium 
hypochlorite,  placed  in  sterilized,  cotton-plugged  test  tubes,  and  left 
in  the  ice  box  for  seven  days.  At  the  end  of  that  time  they  were  cut 
»  into  pieces  with  a  flamed  scalpel  and  some  of  the  sterilized  muck  filtrate 
was  added,  after  which  the  roots  were  placed  in  the  incubator  for  six 
hours.  Before  making  mounts  to  examine  the  material,  a  loopful  of  the 
filtrate  was  transferred  to  each  of  two  petri  dishes,  which  were  then  poured 
with  nutrient  agar.  This  was  done  in  order  to  determine  with  certainty 
whether  or  not  bacteria  were  present.  Germination  was  fully  as  good 
when  the  bacteria  were  not  present  as  when  they  were.  This  is  in  direct 


446  BULLETIN  387 

opposition  to  Pinoy's  (1905)  statement  that  there  is  no  development 
when  the  spores  are  not  accompanied  by  a  coccus. 

Diseased  cabbage  roots  were  disinfected  with  either  mercuric  chloride 
or  calcium  hypochlorite;  if  with  the  former,  they  were  then  rinsed  three 
times  carefully  in  different  tubes  of  sterilized  distilled  water;  if  with 
the  latter,  they  were  rinsed  in  muck-soil  filtrate,  which  is  acid  and  tends 
to  neutralize  any  of  the  calcium  compounds  that  might  adhere  to  the  roots 
and  retard  germination  of  the  spores.  All  the  roots  were  then  either 
transferred  to  tubes  of  nutrient  agar  slants  or  embedded  in  agar  in  petri 
dishes.  If  at  the  end  of  a  week  they  showed  no  signs  of  contamination, 
those  in  the  petri  dishes  were  placed  on  agar  slants,  after  which  all  the 
roots  were  minced  and  left  for  another  week  in  order  to  make  sure  that 
no  bacteria  were  in  the  roots  and  had  been  liberated  by  the  mincing. 

Seeds  of  the  cabbage  were  sterilized  by  the  same  method  as  was 
employed  for  the  roots,  but  they  were  not  rinsed  in  sterilized  water  when 
calcium  hypochlorite  was  used.  The  seeds  were  planted  in  nutrient 
agar  in  petri  dishes  and  the  young  plants  were  permitted  to  develop 
until  they  were  free  of  the  old  seed  coats.  They  were  then  placed  in 
the  tubes  with  the  minced  roots  that  showed  no  bacterial  colonies,  and 
a  sufficient  amount  of  the  sterilized  muck  filtrate  was  added  to  insure 
spore  germination  but  not  enough  to  injure  the  small  seedlings. 

This  process,  tho  complicated  and  long,  seems  to  fulfill  all  the  require- 
ments that  carefulness  demands;  and  in  the  three  series  tried,  from  five 
to  twenty  per  cent  of  the  cultures  were  free  from  bacteria.  The  chief 
difficulty  lies  in  the  fact  that  there  is  such  a  narrow  margin  between  spore 
formation  and  bacterial  invasion  that  it  is  hard  to  select  swellings  which 
are  neither  too  young  to  contain  mature  spores  nor  yet  so  old  that  bacteria 
have  entered.  One  objection  to  the  experiment  is  obvious.  There  is 
no  way  of  determining  contaminations  except  by  the  absence  of  colonies 
on  the  agar  where  the  roots  have  been  minced  and  on  which  the  seedlings 
grow.  Yet  it  seems  hardly  possible  that  bacteria  can  be  present  thruout 
all  these  operations  and  not  come  into  contact  with  the  medium.  Besides, 
where  no  bacterial  colonies  appear,  the  plants  grow  more  vigorously, 
produce  larger  roots,  and  show  infection  sooner  than  in  the  contaminated 
tubes.  Swellings  apparent  to  the  naked  eye  were  formed  at  the  end 
of  five  days  the  first  time  the  experiment  was  tried.  When  the  plants 
were  fixed,  sectioned,  and  stained,  they  showed  amoebae  in  the  cortex 
as  well  as  in  a  large  number  of  root  hairs ;  all  of  which  tends  to  discount 
Pinoy's  (1905)  belief  that  there  is  no  development  of  the  parasites  with- 
out a  concomitant  bacterium. 

Pinoy  based  his  conclusions  in  part  on  the  evidence  presented  by  stained 
sections.  Apparently  he  studied  sections  of  large  roots,  since  the  roots 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS 


447 


that  he  received  from  Mangin  were  evidently  eight  or  more  centimeters 
in  diameter.  The  writer  was  unable  to  procure  thionine,  the  stain  that 
Pinoy  used,  but  he  tried  both  Ziehl's  carbol  fuchsin  and  Kuehne's 
carbol  methylene  blue,  which  have  always  given  good  results  in  staining 
parasitic  bacteria  in  other  tissue.  Parts  of  small,  slightly  swollen  roots, 
as  well  as  pieces  of  larger  roots  (of  which  some  were  still  normal  in  color 
and  others  had  begun  to  turn  black),  were  fixed  in  Carney's  fluid,  con- 
sisting of  glacial  acetic  acid  and  alcohol.  The  small,  slightly  swollen 
roots  after  staining  showed  no  signs  of  bacteria.  Pinoy  (1905)  states 


FlG.    1 10.       SAPROPHYTIC    ORGANISMS    IN    DISEASED    TISSUE 

A,  Partly  corroded  starch  grains  between  the  amoebae,  the  refractive  hila  being  the  only 
visible  part  in  some  of  them;  B,  bacteria  in  a  cell  of  diseased  tissue;  C,  mycelium  of  a  sapro- 
phytic  fungus  in  darkened  diseased  tissue.  X  800 

that  cocci  appear  as  very  refractive  bodies  among  the  amoebae.  In  this 
experiment,  the  hila  of  partly  corroded  starch  grains  (fig.  no,  A)  appeared 
in  several  instances  as  spherical,  brightly  stained  bodies;  but  they  could 
hardly  be  mistaken  for  an  organism,  as  the  same  effect  is  shown  in  healthy 
cells  in  which  entire  starch  grains  may  be  seen. 

The  older,  diseased  tissue  that  has  not  yet  turned  dark  presents  a  some- 
what different  appearance  from  that  of  the  youngest  swellings.  The  epi- 
dermal cells  are  torn  in  many  places,  and  rod-shaped  bacteria  (fig.  no,  B) 
are  found  both  within  and  between  the  cells.  Many  of  these  cells  show 
broken  passages  in  the  walls  where  the  organism  could  easily  have  entered. 


448  BULLETIN  387 

In  a  blackened  root  the  only  additional  change  that  can  be  recognized 
is  the  presence  of  hyphae.  This  blackness  is  almost  a  true  criterion  of 
the  effects  of  a  fungus,  for  the  bacteria  seldom,  if  ever,  produce  any 
pronounced  discoloration  (fig.  no,  c). 

It  is  not  altogether  a  new  phenomenon  to  find  other  organisms  following 
parasitic  slime  molds.  For  example,  the  earlier  writers  who  described 
Sorosphaera  Veronica  regarded  it  as  a  rust  because  of  the  mycelial  threads 
which,  according  to  these  investigators,  are  constantly  present.  Maire 
and  Tison  (1909)  prove  with  but  little  difficulty  that  the  fungi  are  merely 
saprophytic  attendants.  The  case  is  almost  identical  with  that  which 
Schwartz  (1914)  cites  for  species  of  Ligniera  with  which  typical  mycorrhiza 
are  continual  associates. 

It  has  been  shown  that  'non-parasitic  myxomycetes  undoubtedly  make 
use  of  bacteria.  It  seems,  therefore,  altogether  reasonable  that  when 
a  facultative  saprophyte  is  grown  under  conditions  to  which  Spongospora 
subterranea  was  subjected  by  Kunkel  (1915),  it  will  assume  the  habits 
of  a  saprophyte.  As  far  as  this  discussion  is  concerned,  the  only  question 
is  whether  Spongospora  subterranea  still  utilizes  bacteria  when  in  the 
potato  tuber. 

Objection  may  be  found  to  each  of  the  above  experiments  taken  alone. 
When  considered  together  they  cover  the  subject  thoroly  enough,  and 
coincide  so  fully  in  their  results  that  it  seems  logical  to  draw  the  con- 
clusion that  Plasmodiophora  Brassicae  has  no  need  for  the  bacteria  and 
that  the  latter  are  merely  attendant  saprophytic  forms  which  incidentally 
help  to  set  free  the  spores  of  the  parasite.  Only  two  factors  favor  Pinoy's 
theory.  One  is  the  presence  of  bacteria  in  most  roots  in  which  any  con- 
siderable swelling  has  taken  place ;  the  other,  the  fact  that  there  is  a  smaller 
number  of  different  species  of  organisms  present  than  might  have  been 
expected.  Almost  invariably  the  rod-shaped  bacterium  forming  opalescent 
colonies  on  nutrient  agar  was  the  only  one  isolated.  The  facts,  however, 
that  spores  can  germinate  in  sterilized  media,  that  infection  can  occur 
on  seedlings  in  test  tubes  on  nutrient  agar  where  no  bacterial  colonies 
are  present,  and  that  recently  infected  roots  never  show  bacteria  either 
when  tested  in  culture  or  under  the  microscope  after  staining,  would 
seem  to  offset  any  evidence  that  heretofore  has  been  adduced  to  the 
contrary.  Therefore  it  seems  evident  that  Plasmodiophora  Brassicae  is 
an  obligate  parasite,  and,  as  such,  needs  no  other  food  supply  than  that 
furnished  by  its  host. 

SUMMARY 

Neither  the  motility  of  swarm-spores  nor  the  action  of  winds  is  an 
important  factor  in  the  dissemination  of  Plasmodiophora  Brassicae. 


STTDIES  ox  CLUBROOT  or  CRUCIFEROUS  PLANTS  449 

Spores  germinate  better  after  a  slight  rest  period  and  in  such  a  medium 
as  muck-soil  filtrate.  Each  spore  produces  one  swarm-spore,  which, 
if  not  supplied  with  a  host,  develops  no  further. 

It  is  difficult  to  stain  the  flagella  of  swarm-spores,  but  if  they  are  first 
killed  instantly  with  fumes  of  osmic  acid  fairly  good  mounts  can  be 
obtained. 

Penetration  takes  place  thru  the  wall  of  the  root  hair  while  the  organism 
is  in  a  uninucleate  stage.  The  root  hair  at  once  shows  hypertrophy. 
The  amceba  increases  in  size  as  it  passes  rootward,  and  finally,  by  direct 
cell- wall  penetration  as  well  as  by  the  division  of  the  host  cells,  the  patho- 
gene  is  distributed  thruout  the  cortical  tissue. 

The  spores  are  not  always  formed  by  simultaneous  vacuolar  divisions 
of  the  amoebas,  there  being  cases  in  which  they  are  produced  by  successive 
divisions  while  the  adjoining  amcebas  may  still  be  in  the  nuclear  stage. 

Aside  from  Plasmodiophora  Brassicae,  there  is  often  present  another 
organism,  which  causes  no  hypertrophy  and  which  is  probably  Olpidium 
Brassicae  (Wor.)  Dang. 

In  the  experiments  to  determine  the  relation  of  bacteria  to  Plasmo- 
diophora Brassicae,  a  large  number  of  isolations  were  attempted,  diseased 
tissues  of  all  stages  were  stained,  spores  were  germinated  in  sterilized 
media,  and  infections  were  secured  in  test  tubes  under  aseptic  conditions. 
All  this  points  to  the  fact  that  the  bacteria  do  not  enter  the  host  as  soon 
as  the  slime  mold  does,  but  follow  only  after  there  has  been  enough  enlarge- 
ment of  tissues  to  rupture  the  epidermis.  Consequently  the  bacteria 
can  be  of  no  vital  importance  in  the  nutrition  of  the  parasite. 


STUDIES  ON  CLUBROOT  OF  CRUCIFEROUS  PLANTS  451 


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Pamphlet 

Binder 

Gaylord  Bros..  Inc. 
Stockton,  Calif. 

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